This invention relates to the field of clinical chemistry and, more particularly, to an in vitro method for the rapid separation of the MM and MB creatine kinase isoenzymes in blood plasma or serum and for determining the relative activities of the separated isoenzymes.
Three serum creatine phosphokinase (CPK) isoenzymes (MM, MB and BB) have been recognized in tissue extracts and plasma. Myocardium is richly endowed with the MB CPK isoenzyme. Accordingly, increased serum MB CPK activity reflects myocardial injury.
Plasma from normal subjects contains mainly the MM isoenzyme; the MB form accounts for less than 5% of the total plasma activity and activity attributable to BB is negligible, Am. J. Cardiol. 33, 650 (1974). MB activity increases after myocardial infarction, and the magnitude of the increase appears to reflect the extent of myocardial injury. No increase in MB activity is observed after intramuscular injections or after operations not involving the heart. Because of its relative specificity as an index of myocardial damage, a rapid, quantitative assay of MB activity would be particularly useful.
In the prior art, the methods employed to measure the activities of these isoenzymes have been based on electrophoretic (Am. J. Cardiol. 33, 650 (1974) or column chromatographic (Clin. Chem. Acta. 38, 285 (1972)) procedures, which are somewhat complex for routine clinical use. Also in the prior art is a procedure (Clin. Chem. 20, 36 (1974)) utilizing a gel (marketed under the trade designation "DEAE Sephadex A50" by Pharmacia) which suffers from numerous shortcomings. This procedure, for example, entails the handling of small amounts of moist gel and tedious and repeated centrifugations to remove residual MM isoenzyme before desorbing the MB isoenzyme. Further, the procedure requires the production of columns that yield acceptably fast flow rates, stepwise elution, and fractionated collection. Also, one milliliter of plasma is required for each determination.
Thus, while recent advances have facilitated detection of CPK isoenzymes, available techniques have unavoidable quantitative and procedural limitations. There has remained, therefore, a need for a simple, rapid procedure for the quantitation of creatine kinase isoenzymes in blood plasma or serum.